Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance

The failure of metabolic tissues to appropriately respond to insulin (“insulin resistance”) is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

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No human participants were involved in the study.
No human participants were involved in the study.
No human participants were involved in the study.
No human participants were involved in the study.
Sample size was based off our previously published study of insulin signalling in 3T3-Ll adipocyte cells (Humphrey et al. 2013).
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All replicates (n's, denoted in each Figure legend, and plotted as individual data points on relevant graphs) were independent biological replicates. All replication experiments were successful.
Mouse studies were randomized (non-blinded). Liters of mice (comprising n = 6 animals) were allocated to one of three diet regimes (CHOW, HFD, REVERSAL) randomly. After diet completion, litter mates were randomly assigned to one of the two treatment conditions (insulin OR vehicle). Treatments were randomized to dishes/wells for all cell culture studies.
Blinding was not performed for animal studies as it was visually obvious which animals were CHOW or HFD treated due to size differences in both the animals and the volume of fat tissue recovered from each animal.